The overall objective of this research is to gain greater insight into the structure, mechanism of action and metabolic control of acyl-CoA dehydrogenases. Inactive derivatives of the pig kidney acyl-CoA dehydrogenase with iodoacetic acid and several dione reagents are under investigation to identify catalytically essential amino acid residues. Two mechanism based inhibitors of the enzyme, methylenecylcopropylacetyl-CoA and 3,4-pentadienoyl-CoA, are under investigation and preliminary results are suggestive of a mechanism of substrate dehydrogenation involving carbanion formation. The mechanism of stabilization of the red, anionic, seiquinone by acyl-CoA derivatives is under investigation. The results should aid in the interpretation of the mechanism of action of these acyl-CoA dehydrogenases. We will extend our preliminary studies on the reconstitution of pig kidney general acyl-CoA dehydrogenase using ring modified flavin analogs. Efforts will continue to develop a satisfactory purification scheme for pig kidney electron transferring flavoprotein.